Prenatal ethanol exposure is the leading known cause of mental retardation. Growing evidence suggests that excessive cell death is a major component of the pathogenesis of ethanol-induced birth defects. However, there is a fundamental gap in understanding how ethanol leads to apoptotic cell death in embryos. MicroRNAs (miRNAs) are a recently discovered class of small 18-23 nucleotide non-coding RNA that have been implicated in the regulation of genes involved in apoptosis. We have recently discovered that miR-34a, a pro-apoptotic miRNA and miR-125b, an anti-apoptotic miRNA are involved in ethanol-induced apoptosis in neural crest cells (NCCs). Our long-term goal is directed toward the development of effective strategies against ethanol's teratogenesis; strategies based on prevention of ethanol-induced apoptosis through targeting specific pathways involved in apoptosis. The overall objective of this particular proposal is to establish miRNA as a feasible target for the prevention of ethanol-induced apoptosis and teratogenesis. The central hypothesis to be tested is that miR-34a and miR-125b modulate ethanol-induced apoptosis in NCCs by the regulation of p53 and Bcl2 signaling pathways and that the inhibition of miR-34a or overexpression of miR-125b can prevent ethanol-induced teratogenesis. Our hypothesis has been formulated on the basis of strong preliminary data produced in our laboratory. To test our hypothesis, the following specific aims will be addressed: Aim1: To characterize the role of miR-34a and miR-125b in ethanol-induced apoptosis in NCCs. We will determine the effects of ethanol on miR-34a and miR-125b expression in NCCs and in mouse embryos, and the involvement of miR-34a and miR-125b in ethanol-induced apoptosis. Aim2: To test the hypothesis that miR-34a and miR- 125b modulate ethanol-induced apoptosis in NCCs by the regulation of p53 and Bcl2 pathways. This will be accomplished by determining the role of miR-34a in ethanol-induced activation of p53 and suppression of Bcl2 signaling and the effects of down-regulation of miR-125b by ethanol on p53 and Bcl2 signaling in ethanol- exposed NCCs. Aim3: To test the hypothesis that modulation of miR-34a and miR-125b represents a novel therapeutic strategy for preventing ethanol-induced teratogenesis. We will determine whether knockdown of miR-34a or overexpression of miR-125b diminishes ethanol-induced malformations in mouse embryos. The proposed work is innovative, because it focuses on a novel approach, targeting miRNAs involved in apoptosis, to preventing ethanol-induced teratogenesis. The theoretical concept described in this application is also highly innovative because this is the first study attempting to prevent ethanol-induced apoptosis and teratogenesis specifically through the newly recognized actions of miR-34a and miR-125b in apoptosis. The results from this study will be significant, because the insights gained by the accomplishment of these aims will help in elucidating the role of miRNAs in modulating ethanol-induced teratogenesis. They are also expected to yield strategies for preventing ethanol's teratogenesis and to fundamentally advance the field of FASD research.